CN116444447A - 一种sos1和hdac双靶点喹唑啉羟肟酸化合物及其制法和应用 - Google Patents
一种sos1和hdac双靶点喹唑啉羟肟酸化合物及其制法和应用 Download PDFInfo
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- CN116444447A CN116444447A CN202310721093.6A CN202310721093A CN116444447A CN 116444447 A CN116444447 A CN 116444447A CN 202310721093 A CN202310721093 A CN 202310721093A CN 116444447 A CN116444447 A CN 116444447A
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Abstract
本发明公开了一种SOS1和HDAC双靶点喹唑啉羟肟酸化合物及其制法和应用，还公开了一种药物组合物，由上述双靶点化合物或其立体异构体、或其药学上可接受的盐和一种或多种药学上可接受的载体和/或辅料制备而成的制剂，上述化合物具有SOS1和HDAC双重抑制活性，能够抑制肿瘤细胞增殖、促进肿瘤细胞凋亡，显示出优异的体内外抗肿瘤活性，具有更好的成药性前景。
Description
技术领域
本发明涉及一种SOS1和HDAC双靶点喹唑啉羟肟酸化合物，尤其涉及一种SOS1和HDAC双靶点喹唑啉羟肟酸化合物及其制法和应用。
背景技术
宫颈癌是全球女性最常见的恶性肿瘤之一，其早期症状不明显，潜伏期长，多数患者就诊时已处于中晚期，中晚期宫颈癌患者的5年生存率较低，Ⅲ期及以上患者的5年生存率仅为40%-50%，尤其对于转移性和复发性宫颈癌患者来说，其1年生存率仅10-20%，局势不容乐观。目前早期宫颈癌患者采用手术或放疗能够取得较好的疗效，但对于中晚期宫颈癌患者来说，手术操作难度较大，化疗仍是标准疗法，但传统化疗药物的毒副作用及耐药性问题往往使得化疗效果难以达到预期。
SOS1（son of SH-4venless 1）是KRAS的关键核苷酸交换因子，可将GDP结合的KRAS（非活性）转化为GTP结合的KRAS（活性形式）形式；SOS1目前被证明在宫颈癌细胞中过度表达。然而，目前为止仅有一个SOS1小分子抑制BI 1701963进入了临床研究阶段，且该小分子抑制剂的两项临床试验（NCT04835714，NCT04627142）均因产生严重毒性而被宣布终止。迄今为止，市场上还没有针对SOS1的可用抑制剂。
组蛋白去乙酰化酶（histone deacetylases，HDAC）是一类重要的表观遗传酶，通过从组蛋白和非组蛋白中去除乙酰基来调节基因表达，从而参与多个细胞过程，包括细胞周期进程、细胞凋亡、增殖和分化，现有研究表明HDAC的表达水平增加，如HDAC1、HDAC2和HDAC8，会影响宫颈癌的进展。一些HDAC抑制剂已获得FDA批准，例如伏立诺他（vorinostat，SAHA）、罗米地辛（romidepsin）和贝利司他（belinostat）。SAHA是第一个临床批准用于治疗皮肤T细胞淋巴瘤的泛HDACi，然而，它的毒副作用，如血液学和胃肠道毒性，以及耐药性问题限制了临床疗效与应用。
联合给药已被广泛用于由多种信号通路异常引起的复杂性疾病的治疗，相对于单靶点给药联合治疗往往具有更好的抗肿瘤疗效。然而联合给药往往会存在下列问题：联合给药时更易发生急性毒性和迟发性毒性可能更高，特别是在联合使用选择性不强的药物时；由于联合给药涉及多种药物，需要考虑药物的比例和剂量问题，用药方式往往比较复杂，易导致患者依从性差；联合给药可能会导致药物在体内代谢中相互干扰，无法准确预测其药代动力学性质；联合给药可能会出现由药物—药物相互作用引起的不良反应，此外联合给药的药效学特征也难以准确预测。
发明内容
发明目的：本发明的目的是提供一种疗效好且减少耐药性和毒性的SOS1和HDAC双靶点喹唑啉羟肟酸化合物及其制法与应用。
技术方案：本发明所述的一种SOS1和HDAC双靶点喹唑啉羟肟酸化合物或其立体异构体、或其药学上可接受的盐，所述化合物为结构式如式（I）所示的化合物：
。
上述化合物的制备方法包括如下步骤：
（1）将原料1溶于有机溶剂，冰浴下加入浓硫酸，回流搅拌分离纯化得到中间体2；将中间体2溶于乙醇，加入铁粉和饱和氯化铵溶液，反应后分离纯化得到中间体3；将中间体3与乙腈混合，加入盐酸二氧六环溶液，反应后分离纯化得到中间体4；
（2）将中间体4溶于四氢呋喃、二甲基甲酰胺和甲醇的混合溶液，依次加入碳酸铯和7-溴庚酸乙酯，加热反应分离纯化得到中间体5；将中间体5、2,4,6-三异丙基苯磺酰氯以及4-二甲氨基吡啶溶于二氯甲烷和三乙胺的混合液，室温搅拌过夜，随后加入2,4,6-三异丙基苯磺酰氯继续反应，分离纯化得到中间体6；将中间体6与(R)-1-(3-(三氟甲基)苯基)乙胺混悬于二甲基亚砜和三乙胺的混合液，反应分离纯化得到中间体7；
（3）取盐酸羟胺溶于甲醇，搅拌，加入氢氧化钾，冰浴下搅拌反应，抽滤，收集滤液；将中间体7在冰浴下溶解于上述溶液，室温搅拌至反应完全，分离纯化得到SOS1和HDAC双靶点喹唑啉羟肟酸化合物；
。
优选的，步骤（1）具体为：将原料1溶于甲醇，冰浴下加入浓硫酸，回流搅拌至反应产物不再生成，分离纯化得到中间体2；将中间体2溶于乙醇，加入铁粉；加入与乙醇等体积的饱和氯化铵溶液。室温反应4 h，反应至停止放热，分离纯化得到中间体3；将中间体3与乙腈加至圆底瓶，加入盐酸二氧六环溶液，加热反应至原料消失，冷却至室温后产物析出，分离纯化得到中间体4。
优选的，步骤（2）具体为：将中间体4溶于四氢呋喃：二甲基甲酰胺：甲醇的混合溶液，依次加入碳酸铯和7-溴庚酸乙酯，加热反应，分离纯化得到中间体5；将中间体5、2,4,6-三异丙基苯磺酰氯以及4-二甲氨基吡啶溶于二氯甲烷和三乙胺的混合液，室温搅拌过夜，随后加入2,4,6-三异丙基苯磺酰氯继续反应至产物不再生成，分离纯化得到中间体6；将中间体6与(R)-1-(3-(三氟甲基)苯基)乙胺混悬于二甲基亚砜和三乙胺的混合液。加热反应至反应完毕，分离纯化得到中间体7。
优选的，步骤（3）具体为：取盐酸羟胺溶于甲醇，搅拌，加入氢氧化钾，冰浴下搅拌反应，抽滤，收集滤液；将中间体7在冰浴下溶解于上述溶液，室温搅拌至反应完全，分离纯化得到SOS1和HDAC双靶点喹唑啉羟肟酸化合物。
本发明还公开了一种药物组合物，由式（I）所示的双靶点化合物或其立体异构体、或其药学上可接受的盐和一种或多种药学上可接受的载体和/或辅料制备而成的制剂。
优选的，所述药学上可接受的载体和/或辅料包括稀释剂、粘合剂、表面活性剂、致湿剂、润滑剂、填充剂、崩解剂、着色剂、助流剂、稳定剂、助悬剂、缓冲剂、乳化剂、成粒剂、抗粘着剂、胶凝剂、吸收延迟剂、溶解抑制剂、增强剂、吸附剂、螯合剂、防腐剂、着色剂、矫味剂和甜味剂。
优选的，所述制剂为片剂、胶囊剂、口服液、注射剂、冻干粉针剂、透皮剂、气雾剂、固体制剂、脂质体、缓控释制剂、丸剂、栓剂、颗粒剂、散剂、纳米制剂、糖浆剂、酒剂、酊剂、露剂。
上述化合物或其立体异构体、或其药学上可接受的盐，以及上述药物组合物在制备SOS1/HDAC双靶点抑制剂药物中的应用。
上述化合物或其立体异构体、或其药学上可接受的盐，以及上述药物组合物在制备用于抑制RAS信号通路的药物中的应用。
上述化合物或其立体异构体、或其药学上可接受的盐，以及上述药物组合物在制备用于治疗和/或预防肿瘤的药物中的应用。
进一步的，所述肿瘤包括宫颈癌、结肠癌、肺癌、乳腺癌、胃癌、食管癌、前列腺癌、神经胶质瘤、鼻咽瘤、肝癌、卵巢癌或淋巴癌。
有益效果：与现有技术相比，本发明具有如下显著优点：（1）SOS1和HDAC双靶点喹唑啉羟肟酸化合物能够有效抑制细胞增殖、促进细胞凋亡，此外还具有良好的体内抗肿瘤活性且无明显的毒副作用；（2）解决了联合给药存在的药物与药物相互作用、患者依从性差、不可预测的药代动力学和药效学特性等局限性，达到更好的抗肿瘤疗效。
附图说明
图1为实施例1中SH-4的1H NMR 谱图；
图2为实施例1中SH-4的MS谱图；
图3为实施例1中SH-4的HPLC谱图；
图4为实施例4中SH-4抑制SOS1的结果；
图5为实施例5中SH-4抑制HDAC的结果；
图6为实施例6中SH-4促进Hela细胞增殖的结果；
图7为实施例6中SH-4抑制Hela细胞增殖的结果；
图8为实施例7中SH-4抑制Hela细胞凋亡的结果；
图9为实施例8中SH-4对裸鼠体内肿瘤抑制以及对器官毒副作用的影响。
实施方式
下面结合附图对本发明的技术方案作进一步说明。
实施例1
SOS1和HDAC双靶点喹唑啉羟肟酸化合物SH-4的制备：
（1）1.1 2-硝基-5-羟基-4-甲氧基苯甲酸甲酯的合成：
将2-硝基-5-羟基-4-甲氧基苯甲酸(10.0g,46.92mmol,1.0eq) 溶于100mL甲醇，冰浴下加入浓硫酸(5.4mL,98.52mmol,2.1eq)。回流搅拌15h至反应产物不再生成。旋去部分溶剂，加入0.2 N NaOH调节至pH 6后加入EA萃取，水洗，饱和食盐水洗后旋干，得淡黄色固体，为2-硝基-5-羟基-4-甲氧基苯甲酸甲酯 (7.97 g, 74.78%)。1H NMR (300 MHz,DMSO-d 6) δ 10.95 (s, 1H), 7.62 (s, 1H,), 7.08 (s, 1H), 3.91 (s, 3H), 3.80 (s,3H)。
1.2 2-氨基-5-羟基-4-甲氧基苯甲酸甲酯的合成：
将2-硝基-5-羟基-4-甲氧基苯甲酸甲酯(6.0g,26.41mmol,1.0 eq) 溶于50mL乙醇，超声溶解均匀，加入铁粉(5.9g,105.65mmol,4.0eq)。加入与乙醇等体积的饱和氯化铵溶液。室温反应4h，反应放热，放热停止时反应基本完毕。硅藻土助滤去除铁粉，取滤液，旋去乙醇。加入水和EA萃取，有机相水洗，饱和食盐水洗后旋干。得到深黄色固体，为2-氨基-5-羟基-4-甲氧基苯甲酸甲酯(3.95g,75.84%)。1H NMR (300 MHz, DMSO-d 6) δ 8.32 (s,1H), 7.08 (s, 1H), 6.30 (s, 1H), 6.24 (s, 2H), 3.73 (s, 3H), 3.71 (s, 3H); MS(ESI) m/z: 198.1 [M+H]+。
（2）1.3 6-羟基-7-甲氧基-2-甲基喹唑啉-4(3H)-酮的合成：
将2-氨基-5-羟基-4-甲氧基苯甲酸甲酯(1.0g,5.07mmol,1.0eq)与乙腈(2.64mL,50.71mmol,10.0eq) 加入至50 mL圆底瓶，加入4 N HCl的二氧六环溶液(10.14mL,40.57mmol,8.0eq)。60℃反应6h至原料消失(PE:EA=3:1, DCM:MeOH=6:1确认产物生成)。冷却至室温后产物析出，抽滤固体后二氧六环洗，水洗，1 N 碳酸氢钠水溶液洗，水洗，得到白色固体。油泵真空干燥。得到6-羟基-7-甲氧基-2-甲基喹唑啉-4(3H)-酮(0.767g,73.35%)。1H NMR (400 MHz, DMSO-d 6) δ 11.55 (s, 1H), 9.85 (s, 1H), 7.33 (s, 1H),7.00 (s, 1H), 3.87 (s, 3H), 2.28 (s, 3H); MS(ESI) m/z:207.1 [M+H]+。
1.4 7-((7-甲氧基-2-甲基-4-氧代-3,4-二氢喹唑啉-6-基)氧基)庚酸乙酯的合成：
将6-羟基-7-甲氧基-2-甲基喹唑啉-4(3H)-酮(150mg,0.727mmol,1.0eq) 溶于THF:DMF:MeOH=1:1:1的混合溶液，依次加入碳酸铯(284mg,0.873mmol,1.2 eq) 和7-溴庚酸乙酯(155μL,0.800mmol,1.1eq)。65 ℃反应2 h，点板(DCM:MeOH =20:1)反应完全。旋去甲醇和THF后加水和EA萃取，水洗3遍，饱和食盐水洗，无水硫酸钠干燥后旋干。制砂，柱层析分离产物(DCM:MeOH=150:1至40:1)。得到白色固体，为7-((7-甲氧基-2-甲基-4-氧代-3,4-二氢喹唑啉-6-基)氧基)庚酸乙酯(195mg,73.96%)。1H NMR (400 MHz, Chloroform-d) δ11.76 (s, 1H), 7.56 (s, 1H), 7.09 (s, 1H), 4.18 – 4.08 (m, 4H), 3.98 (s, 3H),2.58 (s, 3H), 2.32 (t,J= 7.5 Hz, 2H), 1.98 – 1.84 (m, 2H), 1.73 – 1.64 (m,2H), 1.58 – 1.36 (m, 4H), 1.26 (t,J= 7.1 Hz, 3H);MS(ESI) m/z: 363.2 [M+H]+。
1.5 7-((7-甲氧基-2-甲基-4-(((2,4,6-三异丙基苯基)磺酰基)氧代)喹唑啉-6-基)氧基)庚酸乙酯的合成：
将7-((7-甲氧基-2-甲基-4-氧代-3,4-二氢喹唑啉-6-基)氧基)庚酸乙酯(100mg,0.276mmol,1.0eq)，2,4,6-三异丙基苯磺酰氯(100mg,0.331mmol,1.2eq),DMAP (4mg,27.59μmol,10mol%) 溶于8mL DCM和TEA (116μL,0.828mmol,3.0eq) 的混合液，室温搅拌过夜，加入0.4eq的2,4,6-三异丙基苯磺酰氯继续反应至产物不再生成（DCM:MeOH=20:1），产物点位于磺酰氯和原料之间。DCM/碳酸氢钠萃取，水洗得到粗产物。柱层析得产物（DCM:MeOH=150:1）。完全干燥后得到白色固体，为7-((7-甲氧基-2-甲基-4-(((2,4,6-三异丙基苯基)磺酰基)氧代)喹唑啉-6-基)氧基)庚酸乙酯(95 mg, 54.75%)。1H NMR (300 MHz,Chloroform-d) δ 7.28 (s, 1H), 7.19 (d,J= 2.3 Hz, 3H), 4.33 (h,J= 6.8 Hz, 2H),4.21 – 4.07 (m, 4H), 3.99 (s, 3H), 2.92 (p,J= 6.9 Hz, 1H), 2.49 (s, 3H), 2.34(t,J= 7.5 Hz, 2H), 1.95 (p,J= 6.8 Hz, 2H), 1.71 – 1.62 (m, 2H), 1.65 – 1.36(m, 4H), 1.28 – 1.22 (m, 21H);MS(ESI) m/z: 629.3 [M+H]+。
（3）1.6(R)-7-((7-甲氧基-2-甲基-4-((1-(3-(三氟甲基)苯基)乙基)氨基)喹唑啉-6-基)氧基)庚酸乙酯的合成：
将7-((7-甲氧基-2-甲基-4-(((2,4,6-三异丙基苯基)磺酰基)氧代)喹唑啉-6-基)氧基)庚酸乙酯(150mg,0.238mmol,1.0eq) 与(R)-1-(3-(三氟甲基)苯基)乙胺(58μL,0.358mmol,1.5eq) 混悬于DMSO和TEA (133μL,0.954mmol,4.0eq) 的混合液。90 ℃反应6h至反应完毕(DCM:MeOH:Et3N=12:1:0.1)。利用甲基叔丁基醚/碳酸氢钠萃取。旋干，制砂，柱层析分离(DCM:MeOH=100:1到33:1)。得到无色至淡黄色油状液体，为(R)-7-((7-甲氧基-2-甲基-4-((1-(3-(三氟甲基)苯基)乙基)氨基)喹唑啉-6-基)氧基)庚酸乙酯(43mg,63.34%)。1H NMR (300 MHz, Chloroform-d) δ 7.73 (s, 1H), 7.65 (d,J= 7.8 Hz,1H), 7.55 – 7.48 (m, 1H), 7.47 – 7.40 (m, 1H), 7.14 (s, 1H), 7.01 (s, 1H),5.93 (s, 1H), 5.72 (p,J= 6.7 Hz, 1H), 4.11 (q,J= 7.1 Hz, 2H), 4.04 (t,J= 6.9Hz, 2H), 3.92 (d,J= 2.0 Hz, 3H), 2.54 (d,J= 2.2 Hz, 3H), 2.30 (t,J= 7.3 Hz,2H), 1.86 (q,J= 7.1 Hz, 2H), 1.69 (d,J= 7.0 Hz, 3H), 1.66 – 1.60 (m, 2H),1.46 (d,J= 7.3 Hz, 2H), 1.43 – 1.38 (m, 2H), 1.32 – 1.18 (m, 3H);MS(ESI) m/z:543.3 [M+H]+。
SOS1和HDAC双靶点喹唑啉羟肟酸化合物SH-4的合成：
将934mg盐酸羟胺溶10ml甲醇，0 ℃搅拌5min，加入1.12g KOH，冰浴下搅拌0.5h，抽滤，收集滤液。将(R)-7-((7-甲氧基-2-甲基-4-((1-(3-(三氟甲基)苯基)乙基)氨基)喹唑啉-6-基)氧基)庚酸乙酯(40mg,74.96μmol) 在冰浴下溶解于上述溶液，室温搅拌0.5h。反应完全后，旋干加水，2 N HCl调pH至中性，析出固体，抽滤，晾干或冻干得淡黄色固体，为即为终产物SH-4 (29 mg, 74.32%)。1H NMR (300 MHz, DMSO-d 6) δ 10.40 (s, 1H), 8.72(s, 1H), 8.15 (d,J= 7.8 Hz, 1H), 7.82 (s, 1H), 7.77 (d,J= 6.5 Hz, 1H), 7.71(s, 1H), 7.57 (d,J= 5.9 Hz, 2H), 7.03 (s, 1H), 5.65 (q,J= 7.2 Hz, 1H), 4.08(t,J= 6.6 Hz, 2H), 3.87 (s, 3H), 2.34 (s, 3H), 1.97 (t,J= 6.4 Hz, 2H), 1.80(p,J= 6.8 Hz, 2H), 1.63 (d,J= 7.1 Hz, 3H), 1.59 – 1.50 (m, 2H), 1.45 (q,J=7.4 Hz, 2H), 1.34 (q,J= 9.7, 8.9 Hz, 2H)（图1）；MS(ESI) m/z: 521.2 [M+H]+（图2）；HPLC: 95.124%（Rt = 7.335 min）（图3）（流动相：A相MeOH，B相0.5% HCOOH的水溶液，比例：0-12min A:B=50:50到90:10；12-15 min A:B=90:10到95:5；15-20min A:B=95:5，柱温：40℃，流速：1 mL/min，检测器：254nm）。
实施例2
SH-4对SOS1以及HDACs的抑制作用：首先将SOS1单靶点抑制剂MRTX0902和SH-4化合物溶于缓冲液（10 mM HEPES ( pH 7.4), 5 mM MgCl2, 150 mM NaCl, 1 mM DTT,0.0025% Igepal, 0.05% BSA）中，随后加入SOS1（40 nM，2.5 μL）并在25 ℃下孵育30分钟。然后再加入2.5 μL 缓冲液（80 nM KRASG12C, 1 ng/μL Mab Anti 6His-XL665, 1 ng/μLMab Anti GSH-Eu cryptat），并将混合物在25 ℃下孵育60分钟。在320 nm的激发波长下记录均相时间分辨荧光（HTRF）信号来测定化合物对SOS1的抑制作用。此外，同样利用均相时间分辨荧光技术来测定HDAC单靶点抑制剂伏立诺他以及SE-9化合物对HDACs的抑制作用。结果如表1所示，SH-4对SOS1以及HDAC1、HDAC2、HDAC6和HDAC8均具有显著的抑制作用，且抑制效果优于SOS1单靶点抑制剂MRTX0902和HDAC单靶点抑制剂伏立诺他。
实施例3
SH-4对肿瘤细胞的体外抑制作用：
将人宫颈癌HeLa细胞、人胃癌MKN1细胞、人慢性骨髓白血病K-562细胞、人急性骨髓白血病MOLM-13细胞分别给予不同浓度的SH-4化合物，置37℃，5％CO2 的培养箱中孵育72h，用四甲基偶氮唑盐（MTT）比色法测定化合物对肿瘤细胞的抑制率。结果如表2所示，SH-4对HeLa、MKN1、K-562、以及MOLM-13细胞均具有明显的体外抑制活性。
实施例4
SH-4对细胞SOS1活性的抑制作用：
在用不同浓度的SH-4（0, 0.5, 12.5 μM）处理的Hela细胞中测定RAS-GTP水平，并将EGF作为SOS1激活的阳性对照。如图4所示，不同浓度的SH-4能够降低细胞中RAS-GTP水平，而阳性对照组RAS-GTP水平升高。
为了进一步证实SH-4对SOS1的抑制作用，在相同条件下测量RAS下游效应物p-ERK和p-Akt的水平。如图4所示，p-Akt和p-ERk水平的变化与RAS-GTP的变化一致，即SH-4处理后细胞中p-Akt和p-ERk水平的降低，阳性对照组p-Akt和p-ERk水平升高。这些结果显示，化合物SH-4介导的SOS1抑制可抑制RAS信号传导，并导致下游效应物p-Akt和p-ERK下降。
实施例5
SH-4对细胞HDAC活性的抑制作用：在用不同浓度的SH-4（0, 0.5, 12.5μM）处理的Hela细胞中测定组蛋白H3和H4（I类HDAC底物）以及α-微管蛋白（HDAC6特异性底物）的乙酰化状态，并与FDA批准的HDAC抑制剂SAHA进行了比较。结果显示，0.5μM和12.5μM的SH-4在Hela细胞中诱导α-微管蛋白、组蛋白H3的显著乙酰化，同时在浓度为 12.5μM时诱导了组蛋白H4的显著乙酰化。证实了SH-4是泛HDAC抑制剂。图5中的A，和图5中的B可见，与10μM的SAHA相比，12.5μMSH-4对细胞乙酰化水平的影响更强。图5中的C可见，与DMSO处理的Hela细胞相比，IF分析也显示SH-4处理的Hela细胞中α-微管蛋白乙酰化增加，结果表明SH-4是一种有效的HDAC抑制剂。
实施例6
通过细胞克隆实验考察SH-4对PC-3细胞集落形成能力的影响，以细胞集落形成率与集落形成大小表示细胞独立生存能力，具体过程如下：在6孔板内铺入细胞500个/孔，置于37 ℃恒温培养箱内培养使之贴壁。48h后换入2mL新鲜的完全培养基，再加入不同浓度的SH-4（0, 0.5, 2.5, 12.5 μM），以DMSO作为溶剂配制，再放入培养箱继续培养。之后每3日更换一次培养基。2周后弃去上清液，PBS清洗后加预冷甲醇固定30分钟。然后再加PBS清洗，加入0.1%结晶紫染色液染色30分钟。最后PBS清洗至孔板底部透明无色。静置数天自然干燥，拍照。不同浓度药物处理后的集落形成结果如图6所示，SH-4可以抑制Hela细胞株的克隆形成能力以及细胞活力，并呈浓度依赖性。
使用EdU检测细胞增殖能力：在96孔板中铺入细胞1万个/孔，置于37 ℃恒温培养箱内过夜使之贴壁。次日换入200 μL含不同浓度SH-4（0, 0.5, 2.5, 12.5μM）的完全培养基，以DMSO作为溶剂配制，再放入培养箱继续培养。药物作用24 h后加入含EdU试剂（10μM）的完全培养基，37 ℃孵育2h。PBS清洗5min后加4％多聚甲醛室温固定30min，2mg/mL甘氨酸中和5min，PBS清洗5min后加0.5％ TritonX-100渗透20min，PBS清洗5min。使用kFluor488-azide染色反应液室温避光孵育30min，PBS清洗5min。使用1X Hoechst 33342反应液室温避光孵育30min，PBS清洗后置于荧光显微镜下成像。如图7所示，EdU增殖实验结果也提示SH-4处理可抑制细胞增殖活性，表明SH-4可抑制Hela细胞增殖。
实施例7
本实施例探究SH-4对细胞株凋亡情况的影响。使用梯度浓度的SH-4（0, 0.5,2.5, 12.5μM）处理细胞，以DMSO作为溶剂配制，对细胞进行处理后，用Annexin V-FITC和PI对细胞进行双重标记。如图8中的A所示，SH-4处理可以促进Hela细胞凋亡。进一步地，图8中的B和图8中的C显示检测凋亡蛋白提示SH-4处理可以诱导凋亡蛋白Cleaved PARP和Cleaved caspase 3表达增多，结果表明SH-4可促进Hela细胞凋亡。
实施例8
SH-4对荷人宫颈癌细胞Hela异位移植瘤裸小鼠肿瘤生长的影响：将4-6周龄BALB/C雌性裸小鼠适应环境1周左右，将Hela细胞利用胰酶消化后，1300rpm离心4min，加入5mL新鲜无FBS的DMEM洗一遍后计数，将浓度调整至1×108个细胞/ml，以100μL每只接种至小鼠皮下。待瘤体积长至100mm3后开始给药。每种细胞随机分3组，每组6只，分别为SH-4（1mg/kg,10mg/kg）给药组，溶剂空白对照。接瘤后按组别进行腹腔注射给药，每3日给药1次，每只小鼠给100μL药物（SH-4溶于4％DMSO+1％吐温-80+95％灭菌PBS）或溶剂空白对照（4％DMSO+1％吐温80+95％灭菌PBS），连续3周。实验过程中每4天记录裸小鼠体重和瘤体积，观察裸小鼠生长情况。实验终点取肿瘤组织，测量肿瘤重量并计算抑制率，抑制率＝（对照组瘤体积-加药组瘤体积/对照组瘤体积）×100％。如图9中的A和图9中的B所示，SH-4给药组的肿瘤体积显著减小，但如图9中的C所示并不影响小鼠体重。在低浓度和高浓度下，分别有一个肿瘤和两个肿瘤被SH-4完全根除。此外，测定SH-4对小鼠器官的毒性，图9中的D结果表明，SH-4对小鼠的不同器官（包括心脏、肝脏、脾脏、肺和肾脏）几乎没有毒性。因此SH-4降低了肿瘤生长，且在体内毒性很小。
Claims (7)
1.一种SOS1和HDAC双靶点喹唑啉羟肟酸化合物或其立体异构体、或其药学上可接受的盐，其特征在于，所述化合物为结构式如式（I）所示：
。
2.一种权利要求1所述化合物的制备方法，其特征在于，包括如下步骤：
（1）将原料1溶于有机溶剂，冰浴下加入浓硫酸，回流搅拌分离纯化得到中间体2；将中间体2溶于乙醇，加入铁粉和饱和氯化铵溶液，反应后分离纯化得到中间体3；将中间体3与乙腈混合，加入盐酸二氧六环溶液，反应后分离纯化得到中间体4；
（2）将中间体4溶于四氢呋喃、二甲基甲酰胺和甲醇的混合溶液，依次加入碳酸铯和7-溴庚酸乙酯，加热反应分离纯化得到中间体5；将中间体5、2,4,6-三异丙基苯磺酰氯以及4-二甲氨基吡啶溶于二氯甲烷和三乙胺的混合液，室温搅拌过夜，随后加入2,4,6-三异丙基苯磺酰氯继续反应，分离纯化得到中间体6；将中间体6与(R)-1-(3-(三氟甲基)苯基)乙胺混悬于二甲基亚砜和三乙胺的混合液，反应分离纯化得到中间体7；
（3）取盐酸羟胺溶于甲醇，搅拌，加入氢氧化钾，冰浴下搅拌反应，抽滤，收集滤液；将中间体7在冰浴下溶解于上述溶液，室温搅拌至反应完全，分离纯化得到SOS1和HDAC双靶点喹唑啉羟肟酸化合物；
。
3.一种药物组合物，其特征在于：所述药物组合物由权利要求1中的双靶点化合物或其立体异构体、或其药学上可接受的盐和一种或多种药学上可接受的载体和/或辅料制备而成的制剂。
4.根据权利要求1所述的化合物或其立体异构体、或其药学上可接受的盐，或权利要求3所述的药物组合物在制备SOS1/HDAC双靶点抑制剂药物中的应用。
5.根据权利要求1所述的化合物或其立体异构体、或其药学上可接受的盐，或权利要求3所述的药物组合物在制备用于抑制RAS信号通路的药物中的应用。
6.根据权利要求1所述的化合物或其立体异构体、或其药学上可接受的盐，或权利要求3所述的药物组合物在制备用于治疗和/或预防肿瘤的药物中的应用。
7.根据权利要求6所述的应用，其特征在于，所述肿瘤包括前列腺癌、结肠癌、肺癌、乳腺癌、胃癌、食管癌、宫颈癌、神经胶质瘤、鼻咽瘤、肝癌、卵巢癌或淋巴癌。
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